Requirement for membrane lymphotoxin in natural killer cell development - PubMed (original) (raw)

Requirement for membrane lymphotoxin in natural killer cell development

K Iizuka et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Development of natural killer (NK) cells is thought to depend on interactions between NK progenitors and the bone marrow (BM) microenvironment; however, little is known about the molecular signals involved. Here we show that lymphotoxin (LT) provides an important signal for the development of both NK cells and NK/T cells. LTalpha-/- mice show marked reduction in splenic and BM NK and NK/T cell numbers and dramatically impaired NK and NK/T cell function. Mice deficient in either tumor necrosis factor receptor (TNFR)-I or TNFR-II have normal numbers of NK and NK/T cells, implying that neither of the TNFRs nor soluble LTalpha3 is required for development of these cell types. Reciprocal BM transfers between LTalpha-/- and wild-type mice suggest that close interactions between membrane LT-expressing NK cell precursors and LT-responsive radioresistant stromal cells are necessary for NK cell development. When LT-deficient BM cells are incubated with IL-15, NK cells are formed. In addition, LT-deficient BM cells produce IL-15 after activation. Thus, membrane LT appears to deliver a signal for NK cell development that is either independent of IL-15 or upstream in the IL-15 pathway. These results reveal a novel function for membrane LT in NK and NK/T cell development. They also support a cellular and molecular mechanism by which NK cell precursors themselves deliver essential signals, through the membrane ligand, that induce the microenvironment to promote further NK cell and NK/T cell development.

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Figures

Figure 1

Figure 1

NK cell development and function are severely impaired in C57BL/6 LTα−/− mice. (A) Lower number of NK cells in LTα−/− mice. Splenocytes from 8- to 12-week-old wt and LTα−/− mice were collected and stained for the NK cell marker PK136 (for NK1.1) and the T cell marker CD3. Data from one of five representative experiments are shown. The total numbers of nucleated cells recovered from the spleens and BM of wt and LTα−/− mice were similar. (B) LTα−/− mice failed to reject MHC class I-deficient BM cells in vivo. Irradiated wt and LTα−/− mice (four in each group) were infused with BM cells from β2-microglobulin-deficient donors. BM rejection was measured as described in Materials and Methods. Error bars represent SD of splenic uptake of 125I[UdR] in individual mice. This experiment was repeated once with similar results. (C) Impaired NK cytotoxicity in LTα−/− mice. Fresh splenocytes from C57BL/6 and C57BL/6-LTα−/− mice were used in a standard 51Cr-release assay for natural killing ability in vitro. Error bars represent SD for the six replicates performed for each data point. A similar defect was found with in vivo poly[I]⋅poly[C]-stimulated splenocytes (K.I., data not shown). (D) Lack of NK/T activity in LTα−/− mice. NK/T activity was assessed as production of IL-4 by mice injected with 2 μg of monoclonal anti-CD3 antibody (2C11). For experiments in B and D, the LTα−/− mutation was on a mixed 129/Sv × C57BL/6 background, and wt mice were LTα−/− littermate controls with similar genetic backgrounds.

Figure 2

Figure 2

NK cell development is blocked by treatment of mice with LTβR–Ig. Pregnant mice at gestational day 16 were treated with LTβR–Ig (25 μg) i.p., and each offspring was treated again on day 7 after birth. Splenocytes collected at day 28 after birth were stained for expression of NK1.1. (Lower) Splenocytes from mice treated with LTβR–Ig. (Upper) Control C57BL/6J mice treated with 25 μg of human LFA-1–Ig fusion protein and C57BL/6-RAG-1−/− mice treated with 25 μg of mouse total IgG. (Left) Data from C57BL/6J splenocytes. (Right) Data for C57BL/6-RAG-1−/− splenocytes. Similar data were obtained in four additional experiments with a range of NK cell reduction from 40–80%.

Figure 3

Figure 3

IL-15 can bypass the developmental block of NK cells in LTα−/− mice. (A) When BM cells are cultured in vitro for 7 days with recombinant IL-15, equal numbers of NK1.1+CD3− cells are induced from wt and LTα−/− BM (5.4–5.6 × 106 cells). (B) NK1.1+CD3− cells induced in vitro from LTα−/− (□) and wt (⋄) BM show similar natural killing activity against YAC-1 cells. (C) Expression of IL-15 mRNA in BM cells. Total RNA was prepared from freshly isolated BM cells or BM cells stimulated in vitro with lipopolysaccharide and IFN-γ. IL-15 mRNA was quantitatively analyzed by RNase protection assay. Equivalent amounts of mRNA for glyceraldehyde-3-phosphate dehydrogenase were observed in all four samples.

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